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Comments on Primary Papers and News

The paper from Joseph El Khoury and colleagues presents convincing evidence that the absence of activated microglia is detrimental in the Tg2576 model. On the surface, from our study in J. Neuroscience, one may conclude that microglial activation is harmful. It likely depends on the context of how you are viewing the problem. Early on, microglial activation may be helpful by facilitating clearance of Aβ from brain; in their absence more Aβ accumulates (El Khoury). On the other hand, if Aβ is not cleared and microglia remain activated, this may lead to the chronic neuroinflammation and behavioral deficits that we observed in our model.

Another caveat that we must all recognize is what are the specific features of the models we work with. Each has its own strengths and weaknesses for studying specific aspects of Aβ pathology. For example, the widely used Tg2576 mouse expresses high amounts of Swedish mutant human APP in many cell types, producing high amounts of wild-type Aβ peptides and parenchymal amyloid plaques. The Tg-SwDI mouse expresses low levels of Swedish/Dutch/Iowa mutant human APP only in neurons producing low levels of vasculotropic Dutch/Iowa mutant Aβ peptides and microvascular amyloid deposits. In light of these differences in the models, some variations in results may be attributed to the sites of amyloid deposition and possibly due to differences in microglial responses to wild-type and vasculotropic mutant Aβ peptides and amyloid deposits.

El Khoury et al. have produced a dataset that adds to those indicating a beneficial role for monocytic phagocytes (either activated microglia or hematogenous macrophages) with respect to the development of Alzheimer-related pathology. Some data have indicated that inflammation-related events elaborated by microglia contribute to AD pathology. This includes the overexpression of interleukin-1-β in APP-transgenic mouse models of AD, as well as attenuation of Aβ accumulation in these mice by anti-inflammatory agents such as ibuprofen and, more recently, minocycline (see Fan et al., 2007). But beginning with paradigms in which such mice are immunized against Aβ, increasing evidence has suggested that monocyte-derived cells can help to clear Aβ from the brain through phagocytosis and/or expression of Aβ-degrading proteases. For instance, Morgan and colleagues have shown that injection of the powerful inflammatory agent lipopolysaccharide into APP-transgenic mice results in Aβ clearance (DiCarlo et al., 2006), and the clearance or prevention of Aβ deposits in immunized mice is associated with some signs that microglia are more active.

An important question has been whether these beneficial roles of monocytic phagocytes are operative in the basal condition (and eventually overwhelmed in the development of disease) or are instead induced only by extraordinary manipulation, such as immunization or injections of lipopolysaccharide. El Khoury’s approach was to remove or reduce a chemokine receptor (CCR2) responsible for trafficking microglia and/or peripheral macrophages to sites of inflammation, which would include amyloid plaques in the APP transgenic mice. The resulting increase in Aβ accumulation (both soluble and deposits), coupled with an absence of the accumulation of monocytic phagocytes that normally arises in APP transgenics, suggests that monocyte-derived cells tonically participate in the removal of Aβ; microglia from the CCR2-knockout mice still reacted to Aβ in culture. This specific requirement for chemotaxis, then, is consistent with recent studies showing the homing of bone marrow-derived monocytic cells to plaques in APP transgenics (Simard et al., 2006). Microglia are so extensively distributed throughout the cortex that one should imagine they scarcely need to migrate if they were the primary mediators of Aβ clearance.

Of course, the caveat that an APP transgenic mouse is not a human with AD goes without saying. And that may be most relevant to the interpretation of what happens downstream of Aβ clearance. El Khoury et al. reported a decrease in lifespan in the CCR2-knockout animals, but this may have been due to cerebrovascular hemorrhage. It is possible that well-intentioned clearance of Aβ, regardless of how successful, may produce byproducts that interfere with neurophysiology. To wit, the application of the anti-inflammatory antibiotic minocycline by Fan et al. protected against behavioral deficits in APP transgenic mice without altering Aβ levels or deposition, and ibuprofen treatment is associated with a decrease in a marker of apoptosis per plaque rather than a reduction in plaques themselves (Lim et al., 2001). Thus, strategies aimed at optimizing the impact of inflammatory processes or monocytic phagocytes on AD pathogenesis should take into account the potential requirement of a balance between the benefits of Aβ clearance and the maladaptive consequences of inflammatory sequelae on neuronal function and viability.

It is somewhat unfortunate that El Khoury et al. utilized an APP-transgenic strain that has a mixed genetic background (SJL x C57BL/6). Aβ deposition is notoriously strain-dependent, with the relevant alleles remaining unknown. Any cross of a mixed background creates the opportunity for genetic variability in the progeny, even in littermates. This concern can be mitigated by analyzing sufficient numbers. El Khoury et al. used as few as three or four animals per group, which seems low except for the fact that techniques were applied which precluded the use of the same animals for some of the techniques (e.g., immunohistochemistry vs. FACS); thus, the true numbers of animals over which dramatic differences were seen was actually six or seven per genotype.

It is odd that an effect was noted by El Khoury et al. in a Ccr2 knockout. Cedric Raines showed in a landmark paper that Ccr2 was so redundant that it made no impact on trafficking of monocyte-related cells in EAE (experimental autoimmune encephalomyelitis).

The report by El Khoury and colleagues shows that recruitment of macrophage-like cells to the brains of Tg2576 mice via Ccr2 plays an important role in limiting AD-like pathology. This is a very interesting finding and extends the work of Stalder et al. (2005), who noted the presence of round, non-process-bearing, macrophage-like cells in APP23 mice with appreciable amyloid deposits.

El Khoury et al. have gone further by establishing that Ccr2-dependent recruitment of microglia/macrophage-like cells is important in limiting progression of cerebral amyloidosis. If taken to the logical endpoint, this would mean that microglia and/or macrophages serve to limit amyloidosis by phagocytosing/clearing amyloid deposits in AD mice in the absence of genetic manipulation (and perhaps something similar may occur in AD patients). However, careful 3D reconstruction of microglia and amyloid in APP23 or Tg2576 mice fails to show this (Stalder et al., 2001; Wegiel et al., 2004).

An alternate explanation is that microglia/macrophages secrete a soluble factor (e.g., a cytokine or protease) that limits cerebral amyloidosis. Yet, the converse—that reactive glia produce acute phase reactants/cytokines such as ApoE, ACT and IL-1 that promote amyloidosis—has been shown (Potter et al., 2001; Nilsson et al., 2001). In light of these reports, what is the authors’ take on the mechanism responsible for their finding?

El Khoury et al. also report that Ccr2 deletion limits the lifespan of Tg2576 animals, and suggest that there is a connection between increased AD-like pathology in Ccr2-deficient Tg2576 mice and their premature death. This conclusion should be taken with caution. Although not often pointed out, Tg2576 mice actually overexpress the mutant human APP transgene in regions other than the brain (for example, peripheral vascular smooth muscle cells and endothelial cells), and it is well-established that transgene-derived Aβ is easily detected systemically in these mice, so early death of Ccr2-deficient Tg2576 mice may be CNS-independent.

When taken together, the studies suggest that “activation” of microglia/macrophages is not simply one phenotype. We have suggested that these innate immune cells may respond with a range of responses from pro-phagocytic/anti-inflammatory to anti-phagocytic/proinflammatory (Town et al., 2005). Understanding the molecular underpinnings of these various responses of microglia/macrophages will likely be key in targeting these cells for therapeutic intervention in neurodegenerative diseases (particularly AD).